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LC-MS/MS Practical Mass Spectrometry Training Track: Understanding the Importance of System

Suitability Testing for Monitoring Instrument Health

P H I L I P S OBOLESKY, P H . D.

U C SAN D I EGO H EALT H SYSTEM

Center for Advanced Laboratory Medicine

MSACL Website Resources

What is a System Suitability Test (SST)?

• Tool for monitoring LC and MS health status– General

– Assay Specific

• Sample injected onto LC-MS instrument before submitting patient sample batch

Acceptable SST Provides Assurance

SST Does Not Assure Everything

Ordering Errors

Collection Errors

Labeling Errors

Transport Issues

Processing & Handling

Storage

Reporting ErrorsPre- and Post-Analytical

Errors Still Apply

What Should be Monitored During a SST Run?

Peak Symmetry

Background Noise

Peak HeightSignal

Retention Time

Signal

1. 2.Retention

TimetR (± 0.2 min)

3.Peak Area

4.Peak Symmetry

± 0.2 min

Example of SST Tracking Data

Why Develop and Run a SST

• Increased Efficiency by Avoiding• Re-extractions

• Re-injections

• QNS reports

• Identify Problems sooner

• Tracking records help to identify• Proper maintenance (column or guard replacement, MS

cleaning etc.)

• Convincing vendor to perform PM when MSMS infusion is “within spec”

SST Troubleshooting Cases

PROBLEM ANALYSIS SOLUTION

Case 1: Importance of Tracking Assay Specific SST Data

• 25-OH Vitamin D SST passes after annual PM (service rep leaves)

• Benzodiazepine SST ran next day and FAILS.

Normal Benzo SST

Benzo SST after PM25-OH VitDSST after PM

Case 1: Important Differences Between the Assays

25-OH Vitamin D Benzodiazepine

1. Column 2.1x75 mm, 2.7 μm particle size, C18, HSS T3, 35°C

2. Isocratic LC method

3. Average Column Pressure ~5000 psi

1. Column 2.1x50 mm, 1.7 μm particle size, C18, BEH, 40°C

2. Gradient LC method

3. Average Column Pressure ~7500 psi

Mobile Phases Identical

Case 1: Resolution & Lessons Learned

• Problem: Leaky Check valve at pressures >7000 psi

• Solution: Replace check valve

• Lessons Learned:1. A single SST on a “within spec” instrument does not mean its okay

for all assays.

2. Run all assay SSTs on an instrument after a PM before letting engineer leave.

Case 2: Spotting Column Degradation using SST Data

Normal Conditions >50 Injections

Case 2: Developing a SST to Assure Acceptable Chromatographic Resolution

1. Make Neat SST solution containing more Oxazepam than Desalkylflurazepam

2. If unacceptable – Change the guard and/or column

Unacceptable Resolution

Overlay 289>140 MRM QC 10ng/mL Desalkylflurazepam detecting the Desalkylflurazepam peak

Acceptable Resolution

289>140 MRM Desalkylflurazepam detecting Oxazepam

289>140 MRM Desalkylflurazepam detecting Desalkylflurazepam

Case 2: Resolution & Lessons Learned

• Problem: Poor chromatographic resolution due to sample buildup

• Solution: Run SST that assures peak resolution acceptable

• Lessons Learned:1. Some post-validation issues can be addressed with SST preventing

revalidation

Case 3: Troubleshooting Blank SST TIC• Blank SST Chromatogram Empty vial?

Was the vial pierced?

Run errors?

Key Points to Remember about SST

1. A single SST on a “within spec” instrument does not mean its okay for all assays.

2. Run all assay SSTs on an instrument after a PM before letting engineer leave.

3. Some post-validation issues can be addressed with SST preventing revalidation

4. Start by checking the obvious when troubleshooting

AcknowledgementsThanks to the staff of the UCSD Toxicology/Mass Spectrometry Laboratory who solved and documented this troubleshooting case:• Judy Stone, MT(ASCP), Ph.D., DABCC

• Josh Akin

•Heather Hochrein

•Dawn Francisco

•Robert Fitzgerald, Ph.D., DABCC

•UCSD Clinical Chemistry Fellows• Imir Metushi, Ph.D., ABCC

• Jacqueline Hubbard, Ph.D.

https://www.surveymonkey.com/r/7WQPWJC

Please let us know what training resources you need

chris.herold@msacl.org jastone@ucsd.edu

Speaker and Presentation Evaluations for Sobolesky/Akin/ Metushi Survey Monkey

Zen and the Art of LC-MS/MS Maintenance Assuring Optimal Instrument Performance

Joshua AkinUC San Diego Health

DisclaimerThis talk should in no way be associated with that great body of factual information relating to orthodox Zen Buddhist practice,

though it is very factual in chromatography and mass spectrometry.

Overview and ObjectivesGoal: Assure optimal and reliable performance of your LC-MS/MS instrument

Understanding the importance of regular maintenance

Develop a checklist to aide in the tasks and intervals required of instrument maintenance

Why is Maintenance so ImportantIrv Gordon’s 1966 Volvo 1800S w/ 3,039,122 miles

"Just follow the owner's manual; it's a piece of machinery and can't take care of itself. Have its scheduled maintenance

completed… and when it makes a funny noise, listen to it."

Monitor gauges

Change oil

Check pressure

Clean Intake

Top-off fluids

Why is Maintenance so Important

LC-MS/MS instruments are no exception

Monitor gauges

Change oil

Top-off fluids

Check pressure

Clean Intake

Maintenance Checklists –A Roadmap to Success

LC-MS/MS instruments are no exception

Where to Begin?CLSI Document C50-A “Mass Spectrometry in the Clinical Laboratory: General Principles and Guidance - Approved Guidelines”

CLSI Document C62-A – “Liquid Chromatography-Mass Spectrometry Methods - Approved Guidelines” No Guidance on

Maintenance!

Where to Begin?CLSI Document GP31-A – “Laboratory Instrument Implementation, Verification, and Maintenance; Approved Guidelines”

◦Provides guidelines on:

◦Establishing procedures

◦Proper documentation

◦Responsibilities

Not Specific to LC-MS/MS Instrumentation!

Where to Begin?Manufacturer Documentation

◦Maintenance schedules

◦Procedures

◦Troubleshooting

For use only as GUIDANCE to prepare a procedure

SPECIFIC to your laboratory

MANUAL!

Before Moving ForwardAdequate source of power/backup (UPS)

Data repository/server

Gases (i.e., nitrogen, argon) are properly supplied

LC-MS/MS grade reagents and ultrapure H20

Service contract – more extensive service

Start with the Basics

1) Month and Year2) Day of the Month3) Frequency4) Tech Initials

Maintenance intervalsDaily

Weekly

Monthly

Yearly

As needed

Daily Maintenance

Is there enough mobile phase for start-up and analysis?

Assure adequate volume for duration of analysis

Can be approximated:

◦ flow rate (ml/min) X length of run (min)= volume needed

• “Drying up” fluidics system can damage pumps

Mobile Phase Reagents

Daily Maintenance

Are the instrument vacuum readings within acceptable range?

Are the turbo pumps operating as expected?

Vacuum Monitoring

0.00E+00

5.00E-05

1.00E-04

1.50E-04

2.00E-04

2.50E-04

1 4 7 1013161922252831 3 6 9 12151821242730

Collision Cell Vacuum

0.00E+00

5.00E-04

1.00E-03

1.50E-03

2.00E-03

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61

Ion Source Vacuum

Tracking longitudinally can give insight into problems

Leak coming from source enclosure

Interruption in the argon supply

Daily Maintenance

Was system suitability performed on this instrument?What column/method was used?

See Phil’s Lecture!

Weekly Maintenance

Were the interface cones properly cleaned?

Cleaning Sample ConesDepends on the degree of contamination

• Regular use:

1:1 MeOH/H20

• Grossly contaminated: 45:45:10 MeOH/H20/ Formic Acid

• 10 min sonication• Blow dry

Weekly Maintenance

Was the computer rebooted and disk cleanup performed?

Reboot removes “Operating Footprint”

◦ Background processes◦ Hoards valuable system resources (usually

• Disk Cleanup frees disk space on HD

– Consult with IT for specific files

Reboot and Disk Cleanup

Weekly Maintenance• Was the oil level

checked on the roughing pumps

• Were the pumps ballasted?

Pump Ballast and OilBallast Valve: purging pump oil of condensate (water/solvent vapors)

◦ Recommended: 15-30 minutes at operating temperature

Ballast Valve

Pump Ballast and OilCheck oil level and note color

◦ Change every 6 months or sooner

Weekly Maintenance

Were LC guard columns replaced?

Guard columnsChange at regular time or injection intervals

Inspect ferrule and replace if needed

Minimize dead-volume by assuring tight connections

Column Guard column

Flow Path

Monthly Maintenance• Is data backed-up

properly?• Are security

updates current?

Data Back-upReal-time Server-based (LIS Intranet, LAN)

PeriodicScope? (full vs. incremental)

Frequency?

Backup media (tape, floppy, disk, etc.)

Windows Security Updates

• Security or Critical updates: malware and security exploits.

• Other updates: typically unrelated to security or enhance functionality.

Friday May 12th, 2017

Key PointsRoutine maintenance is the cornerstone of a happy, healthy LC-MS/MS

Craft a checklist that is suited for your particular lab

Implement a maintenance procedure that assures the reliability and longevity of your instrument.

AcknowledgementsUCSD Toxicology/Mass Spectrometry Laboratory

◦ Rob Fitzgerald, Ph. D., DABCC◦ Judy Stone, MT (ASCP), Ph. D., DABCC◦ Heather Hochrein, MT (ASCP)◦ Dawn Francisco, MT (ASCP)

Questions? Need help?

jakin@ucsd.edu

Thank you!

Choosing the right internal standard for LC-MS/MS

Imir G. Metushi (PhD, DABCC, FAACC)

Director, LabCorp (Endocrine Sciences)

Calabasas CA

Disclosures

Acknowledgments

The following slides include troubleshooting cases documented by LabCorp, Burlington and RTP

Slides are courtesy of Russ Grant and Brian Rappold (LabCorp, Burlington and RTP).

Outline

What is the value of an internal standard in LC-MS/MS

How do we use it

What can go wrong

External Calibration

“known” amounts of analyte

External Calibration Samples

Unknown Test Samples ?

1 5 10 50

External Calibration curve

Unknown Samples

? ? ? ?

Total Efficiency

Analyte must have identical matrix effects and extraction efficiency (i.e., total efficiency) between calibrators and samples for accuracy

External Calibration can demonstrate recovery differences

Hydrolysis (100% Efficiency)

-50% Bias

What is an Internal Standard

An internal standard in analytical chemistry is a chemical substance that is added in a constant amount to samples, the blank and calibration standards in a chemical analysis. This is done to correct for the loss of analyte during sample preparation, injection and ionization.

Source: wikipedia

S L I D E

Internal Standards

UTILITY:

Normalization of recovery differences

Injection variance

Identification of analyte retention time shift

• And peak shape

Normalization of ionization effects between calibrators and samples

One of the most valuable components in LC-MS/MS analytical quality

Retinol D4 Retinol

0.05 0.15 0.25 Time, min

sity, cps

0.05 0.15 0.25 Time, min

sity, cps

0.05 0.15 0.25 Time, min

sity, cps

0.05 0.15 0.25 Time, min

Retinol D4 Retinol

Good IS informs what the analyte retention time and peak shape should be Allows diagnosis of problems

What Does the IS Tell You Qualitatively

Qc1 (injection #12) Qc1 (injection #94)

IS Peak Area Trend: Outliers and Drift

Tecan Liquid handler IS addition Using same 8 tips with aqueous D4-Cortisol

Drift across run observed

IS peak area precision enables outlier detection

With 0.1% BSA (aq) D4-Cortisol solution and Pre-wetted tips (x3) prior to dispensing

IS peak area CV = 8.6%

External Calibration with Internal Standardization “known” amounts

of analyte

Unknown Test Samples ?

Unknown Samples

Total Efficiency

1 5 10 50

? ? ? ?

D[A/IS] = 0

Difference in the matrix effects and extraction efficiency (total efficiency) incurred by the Analyte between calibrators should be identical to the difference incurred by the Internal Standard

DA=DIS

External Calibration with Poor Internal Standardization

+Labeled-IS

50% Bias

External Calibration with Good Internal Standardization

0% Bias

+Labeled-IS

Internal Standards: Needs and issues

NATURE:

Structurally unique (exogenous) – Not observed in samples

Resolved (separated) from analyte(s) by MS, but Co-elutes

Stable labeled isotope (2H, 13C, 15N, 18O) - +3 amu*, pure

Structurally similar (analog) – No recovery or matrix effect correction

Structurally dissimilar - injection check at best?

XIC of +MRM (77 pairs): 150.1/87.1 Da ID: Lysine D3 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Spra... Max. 9.4e5 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26Time, min

D3-Lysine (m/z): 150-87 Tr = 18.97

XIC of +MRM (77 pairs): 150.1/104.1 Da ID: Methionine from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Sp... Max. 3.8e5 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26Time, min

Methionine (m/z): 150 – 104 Tr = 12.57

XIC of +MRM (77 pairs): 153.1/107.1 Da ID: Methionine D3 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turb... Max. 1.6e5 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26Time, min

D3-Methionine (m/z): 153 – 107 Tr = 12.57

XIC of +MRM (77 pairs): 153.1/88.1 Da ID: Glutamic Acid D5 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Tur... Max. 1.9e5 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26Time, min

D5-Glutamic Acid (m/z): 153 – 88 Tr = 13.68

XIC of +MRM (77 pairs): 119.1/73.1 Da ID: Proline D3 from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Spr... Max. 1.1e6 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26Time, min

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

1.15e6

13.87 D3-Proline (m/z): 119-73 Tr = 13.84

XIC of +MRM (77 pairs): 118.1/72.1 Da ID: Valine from Sample 1 (Plasma Mid QC_) of SpecChemQC 030211NPExt.wiff (Turbo Spray) Max. 1.3e6 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26Time, min

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

1.10e6

1.15e6

1.20e6

1.25e6

Structurally Unique in specimens

Valine (m/z):118-72 Tr = 12.89

Internal Standards

NATURE:

Structurally unique (exogenous) – Not observed in samples

Resolved (separated) from analyte(s) by MS, but Co-elutes

Stable labeled isotope (2H, 13C, 15N, 18O) - +3 amu*, pure

Structurally similar (analog) – Limited recovery or ionization effect correction

Structurally dissimilar - injection check at best?

Too many Deuterons can hurt you

D10-Gabapentin 182.2→147.1

Gabapentin 172.2→137.1

XIC of +MRM (4 pairs): 172.200/137.100 Da ID: Gabapentin 1 from Sample 9 (Blank) of ASMS_Gaba_res5.wiff (Turbo Spray) Max. 4.4e4 cps.

0.69 0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84 0.85 0.86 0.87 0.88Time, min

2000.0

4000.0

6000.0

8000.0

Fast LC: Co-elution

XIC of +MRM (4 pairs): 172.200/137.100 Da ID: Gabapentin 1 from Sample 9 (Blank) of ASMS_Gaba_res4.wiff (Turbo Spray) Max. 2.6e4 cps.

1.33 1.34 1.35 1.36 1.37 1.38 1.39 1.40 1.41 1.42 1.43 1.44 1.45 1.46 1.47 1.48 1.49 1.50 1.51 1.52Time, min

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

Slow LC: Separation

1:1 Gabapentin:D10 IS in Clean Urine

D10-Gabapentin IS not Co-eluting

Under-labelled IS

11-Desoxycortisol Concentration (ng/dL) 400 800 1200 1600 2000

alyte Area / IS

Calibration curve non-linear 3-log range for 11-Desoxycortisol

IS only 13C2 labelled (*)

Analyte isotopically contributes to IS -

Solutions:

Truncate to linear range assay neat and pre-dilute samples (two analysis!),

Repeat on dilution > mid point

Increase IS concentration or alternate IS

Clinically acceptable as is

Abnormal (>156ng/dL)

S L I D E

Internal Standard

ADDITION:

FIRST step after mixing/pipetting sample (see Glucoronide IS example)

Solution ideally miscible with sample – to correct analyte recovery

Mixed well prior to extraction – Equilibrated identically to analyte

Internal Standard stability to be checked – In solution and H/D exchange in ion source

Reproducibly added (precise!) to samples, calibrators, QC’s except double blanks

Protein Precipitation – Serotonin Blood vs Serum

Spike & Recovery

Mix and Pipet 100uL Sample

Add 300uL D4-Serotonin IS in Acetonitrile

Mix and centrifuge 10 min each

Transfer to 96-well plate and inject

Internal Standard Equilibration in Samples Reverse Timing Study

S L I D E

Aliquots of sample pool plus IS equilibrated with mixing Decreasing amounts of time prior to extraction in parallel (i.e., reverse timing)

IS 120 min

IS 60 min

IS 30 min

IS 15 min

Equilibration w/ Mixing Extraction

Plateau in A:IS ratio indicates IS has reached binding equilibrium Use double the minimum time!

Serotonin: IS Binding and RBC’s

S L I D E

Equilibration/Mixing Time (min)

S L I D E

Internal Standard

ADDITION:

FIRST step after mixing/pipetting sample (see Glucoronide IS example)

Solution ideally miscible with sample – to correct analyte recovery

Mixed well prior to extraction – Equilibrated identically to analyte

Internal Standard stability to be checked – In solution and H/D exchange in ion source

Reproducibly added (precise!) samples, calibrators QC’s except double blanks – Hard to know with manual pipetting

Analyte observed in IS solution: H/D Exchange

Neat Dopamine D4 solution appeared to contain mostly Dopamine

Labelling is in an active region of the molecule

Deuteriums exchange with Protons in the APCI source

Solution: Used ESI

XIC of +MRM (6 pairs): 154.1/91.1 Da from Sample 12 (IS APCI) of H-D exchange.wiff (Heated Nebulizer) Max. 1.0e5 cps.

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

XIC of +MRM (6 pairs): 158.2/139.0 Da from Sample 10 (IS ESI) of H-D exchange.wiff (Turbo Spray) Max. 3.7e5 cps.

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min

XIC of +MRM (6 pairs): 154.1/91.1 Da from Sample 10 (IS ESI) of H-D exchange.wiff (Turbo Spray) Max. 1133.3 cps.

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min

0.190.09

0.14 0.21 0.30

0.110.05 0.08 0.17 0.56

0.12 0.34 0.58

0.31 0.590.51

0.670.36 0.52 0.71

0.61 0.63 0.660.430.400.38 0.72

XIC of +MRM (6 pairs): 158.2/139.0 Da from Sample 12 (IS APCI) of H-D exchange.wiff (Heated Nebulizer) Max. 2.5e6 cps.

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min

D4-Dopamine IS

1e5 cps

Dopamine

2e6 cps

D4-Dopamine IS

4e5 cps

Dopamine, 1100 cps

IS in Water APCI Source IS in Water ESI source

It can get more complicated: just ask Russ

IS Peak Area vs Index (004102 DHEA)- "Mean" Regression ("No" weighting): mean = 8.11e+004 x (std. dev. = 1.44e+004)

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95Index

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5IS

D5-Phenylalanine Peak Area Response in a run

Final thoughts

S L I D E

Rule 1: Internal standards are NOT a universal accuracy fix.

Rule 2: Ensure adequate binding/equilibration for analyte and IS prior to extraction (time, pH, temperature, mixing)

Rule 3: IS stability during extraction relative to analyte will impact accuracy (e.g., H/D exchange).

Rule 4: IS recovery (and matrix effect for D-labeling) should be identical between calibrators and samples

Rule 5: Don’t abuse the IS – it is literally your best friend

Acknowledgments

Philip Sobolesky at UC San Diego

MSACL Committee

Russ Grant and Brian Rappold (LabCorp Burlington and RTP) for the slides

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